A Capillary Electrophoresis (CE) separation can be performed to determine what affinity, if any, may exist between some xe2x80x9cinterestingxe2x80x9d target molecule and a small molecule ligand. The origin of the ligand can be from natural product samples, synthesized pure compounds in a combinatorial library, or mixtures of compounds. An exemplary method of screening complex biological materials for use in accordance with the present invention is described in Hughes et al., U.S. Pat. No. 5,783,397 (Jul. 21, 1998), the whole of which is incorporated by reference.
Performing an electrophoresis separation is not typically a process that lends itself to automation and application to high throughput screening (HTS) environments except in relatively simple applications like DNA sequencing. Historically, high sample analysis throughput rates are typically achieved by utilizing an array of capillaries which process samples in parallel. There are several disadvantages of using such multiple capillary array in a practical HTS xe2x80x9cfactory environment.xe2x80x9d The cost of producing the intricate capillary array assembly is high. The capillary array temperature can be difficult to control. Time-critical sample preparation steps must be executed for each sample at specific and repeatable times prior to the CE injection and separation event. Using a low temperature process to remove the external polyimide coating to create a sample viewing window in each of the array capillaries (which is mandatory when using capillaries with inside wall surface treatments) can be difficult. The logistics of reprocessing multiple missed samples should a single capillary in the array fail during a run can be complex. For CE systems with multiple capillaries built into a complex and expensive array assembly, if one capillary becomes defective, it is not economically feasible to immediately switch out the whole array. It will be allowed to continue running for a period of time until the number of performing capillaries drops to 90 or 80 percent of the total. In the meantime, special effort must be expended to analyze separately the samples that were to have been processed by the defective channels.
Unlike High Performance Liquid Chromatography (HPLC) separation technology where samples are inserted into the analysis stream with special valving, the volumes of sample liquid are much too small in CE to be handled in this manner. Conventionally, a CE capillary is closely integrated with an immovable ultraviolet (UV) absorbance or laser induced fluorescence (LIF) detector. Because the detector and capillary are stationary, the sample and buffer vials in most instrumentation are designed to be sufficiently mobile to provide the flexibility required for traditional CE methods. Typically, the liquid vials containing the sample, rinsing and running buffers are automatically positioned as specified by the operator""s method program so as to immerse the stationary inlet and outlet ends of the capillary into the required liquids.
A typical CE affinity assay requires integrating the sample preparation process with the action of injecting the prepared sample cocktail into the CE analysis capillary. This requires, by some means, the immersion of the inlet end of the capillary into the container with the prepared sample, creating a pneumatic seal, and applying injection pressure to the contained volume above the sample liquid.
In very short, fast run-time capillary configurations where the capillary inlet can be as close as 6 cm to the detection window, it is not feasible to mechanically isolate the capillary injection point (the capillary inlet end) from the detection window with its associated detector optics. For this reason, existing CE instruments use complex mechanical arrangements to transport each sample vial to the inlet end of the capillary because the capillary must be fixed to the relatively massive detector mechanism.
The present invention relates to a high throughput screening (HTS) capillary electrophoresis (CE) system comprising a plurality of miniaturized mobile detector modules. The detector modules of the invention are transported by a programmable robotic arm assembly to an area of fixed sample wells for analysis. The arm assembly is also capable of integrating sample preparation simultaneously and independently of the CE analysis. The invention achieves high throughput CE assay, which requires a complex series of precise sample preparation steps, resulting in a time critical injection window, followed immediately with CE injection, separation, and data collection processes.
The miniaturized detector modules contain a single capillary precluding any maintenance of an intricate multi-capillary assembly. The operation of a single detector may be paused as soon as any degradation in the electrophoretic performance is observed without affecting the throughput and productivity of the remaining active detectors. This is not only cost-effective but it also reduces the extra expense of sample preparation materials, and the time and effort of repeating the defective sample analysis.
The capillary in the miniaturized detector module is also temperature controlled. Maintaining a controlled capillary temperature results in consistent and accurate separation analysis. Depending on the CE assay, some commercially available capillaries are distinctly temperature sensitive, deleteriously affecting the performance of the capillary. As one embodiment, the detector modules of the invention contain a heat sink to absorb extraneous heat as well as a channel for liquid coolant to circulate, maintaining a constant temperature environment for the capillary. A low temperature process is also used to remove the external polyimide coating on, for example, fused silica capillary for creating a separation detection window.
The detector modules of the CE system of the invention are adapted to include any type of detection, preferably an ultraviolet (UV) absorbance detection or a laser-induced fluorescence (LIF) detection. Other types of detection include, but not limited to, visible light absorbance, fluorescence polarization, conductivity, radioactive, and electrochemical detection. The detector modules are configured to increase sensitivity of detection by customizing the optics assembly for the detectors as well as modifying the internal detection area for decreasing extraneous light energy.
The CE system of the invention is fully programmable and automated by a controller assembly, which is interfaced with the arm assembly and the miniaturized detector modules. The CE system is programmed to handle time-critical sample preparation steps required prior to CE sample injection and separation analysis. Sample preparation and analysis are coordinated to produce HTS CE results by having a single microfluidic pipette on the arm assembly work in concert with a plurality of miniaturized detector modules. After a sample is prepared with the single pipette resource, one detector module can analyze the sample, while the same pipette prepares another sample for analysis by a different detector module. Maximum throughput is established by balancing the sample preparation time with the analysis time for the whole system. For example, if a given assay requires four minutes of analysis time and one minute of sample preparation time, then four detectors would be configured with a single pipette.
Accordingly, such an efficient and versatile CE system of the invention increases throughput of CE assays as well as the sensitivity and repeatability of the assays as more fully described below.